Process for the conversion of echinocandin class of peptides to their c4-homotyrosine monodeoxy analogues

ABSTRACT

The invention relates to a process for the conversion of echinocandin class of peptides to their C4-homotyrosine monodeoxy analogues, particularly mulundocandin to deoxymulundocandin, which consists of a single step selective reduction of C4-htyr (homotyrosine) hydroxyl group of echinocandins to their monodeoxy analogues under neutral conditions without prior protection/deprotection of the equally facile C5-Orn (ornithine) hydroxyl group and purification of the monodeoxy compound from the crude reaction mixture.

FIELD OF THE INVENTION

This invention relates to a process for the conversion of echinocandinclass of peptides of the formula I

wherein W, X, Y, Z, R and R′ are as defined herein below:

W X Y Z R R′ 1. Echino- OH OH OH OH CH₃ Linoleoyl   candin B 2. Pneumo-OH OH OH OH CH₂—CONH₂ 10,12-Di-   candin A₀ methyl- myristoyl 3. Pneumo-H OH OH OH CH₂—CONH₂ 10,12-Di-   candin A₁ methyl- myristoyl 4. Pneumo-OH OH H H CH₂—CONH₂ 10,12-Di-   candin A₂ methyl- myristoyl 5. Pneumo-OH OH OH OH CH₂—CONH₂ 10,12-Di-   candin B₀ methyl- myristoyl 6. Pneumo-OH OH H H CH₂—CONH₂ 10,12-Di-   candin B₂ methyl- myristoyl 7. Pneumo-OH OH OH OH CH₂—CONH₂ 10,12-Di-   candin C₀ methyl- 8. Mulundo- OH OH OHOH H 12-Methyl-   candin tetradec noyl

to their C4-homotyrosine monodeoxy analogues of the formula I wherein W,Y, Z, R and R′ are as defined herein below:

W X Y Z R R′ 1. Deoxyechinocandin B OH H OH OH CH₃ Linoleoyl(Echinocandin C) 2. Deoxypneumocandin A₀ OH H OH OH CH₂—CO—NH₂10,12-Dimethyl- myristoyl 3. Deoxypneumocandin A₁ H H OH OH CH₂—CONH₂10,12-Dimethyl- myristoyl 4. Deoxypneumocandin A₂ OH H H H CH₂—CONH₂10,12-Dimethyl- myristoyl 5. Deoxypneumocandin B₀ OH H OH OH CH₂—CONH₂10,12-Dimethyl- myristoyl 6. Deoxypneumocandin B₂ OH H H H CH₂—CONH₂10,12-Dimethyl- myristoyl 7. Deoxypneumocandin C₀ OH H OH OH CH₂—CONH₂10,12-Dimethyl- myristoyl 8. Deoxymulundocandin OH H OH OH H 12-Methyltetra- decanoyl,

particularly to a process for the conversion of mulundocandin (compoundof the formula II)

to deoxymulundocandin (compound of the formula III)

BACKGROUND OF THE INVENTION

1,3-β-glucan synthesis inhibitors are effective antifungal agentsCandida albicans and also Pneumocystis carini, an opportunistic organismresponsible for an often fatal pneumonitis among HIV patients and otherimmunocompromised hosts. Of all the structural classes of 1,3-β-glucansynthesis inhibitors, only the echinocandins received considerableattention [Ref: J. Med. Chem. 35, 198-200 (1992)]. Echinocandin class ofpeptides are cyclic hexapeptides having a lipophilic side chain.

Several methods for the conversion of echinocandins to the correspondingdeoxy analogues under acidic conditions have been reported [Ref:Tetrahedron Letts., 33, 4529-4532 (1992); U.S. patent application Ser.No. 222,157 dated Apr. 4, 1994]. The above methods involve selectivereduction of C4-htyr (homotyrosine) hydroxyl group of echinocandins totheir monodeoxy analogues with prior protection/deprotection of theequally facile C5-Orn (ornithine) hydroxyl group.

Mulundocandin [J.Antibiotics, 40, 275-280 and 281-289 (1987)] anddeoxymulundocandin [Indian patent No. IN I69830 ; J.Antibiotics. 45,618-623 (1992)] having antifungal properties were isolated fromAspergillus sydowii (Bainier and Sartory) Thom and Church var. Nov.Mulundensis Roy (culture no.HIL Y-30462). Deoxymulundocandin was foundto possess better antifungal activity than mulundocandin. However, theproduction of deoxymulundocandin during the fermentation was 200 timesless than that of mulundocandin.

We have found out by extensive research and experimentation thatechinocandin class of peptides of the formula I may be converted to thecorresponding C4-htyr monodeoxy analogues, particularly mulundocandin todeoxymulundocandin under neutral conditions. Accordingly, the object ofthe present invention is to provide a process for the conversion ofechinocandin class of peptides of the formula I to the correspondingC4-homotyrosin monodeoxy analogues, particularly mulundocandin (compoundof formula II) to deoxymulundocandin (compound of formula III).

SUMMARY OF THE INVENTION

According to the invention, there is provided a process for theconversion of echinocandin class of peptides of the formula I

wherein W, X, Y, Z, R and R′ are as defined herein below:

W X Y Z R R′ 1. Echinocandin B OH OH OH OH CH₃ Linoleoyl 2. PneumocandinA₀ OH OH OH OH CH₂—CO—NH₂ 10,12-Dimethyl- myristoyl 3. Pneumocandin A₁ HOH OH OH CH₂—CO—NH₂ 10,12-Dimethyl- myristoyl 4. Pneumocandin A₂ OH OH HH CH₂—CO—NH₂ 10,12-Dimethyl- myristoyl 5. Pneumocandin B₀ OH OH OH OHCH₂—CO—NH₂ 10,12-Dimethyl- myristoyl 6. Pneumocandin B₂ OH OH H HCH₂—CO—NH₂ 10,12-Dimethyl- myristoyl 7. Pneumocandin C₀ OH OH OH OHCH₂—CO—NH₂ 10,12-Dimethyl- myristoyl 8. Mulundocandin OH OH OH OH H12-Methyl- tetradecanoyl

to the C4-homotyrosine monodeoxy analogues of the formula I, wherein W,X, Y, Z, R and R′ are as defined herein below:

W X Y Z R R′ 1. Deoxyechinocandin B OH H OH OH CH₃ Linoleoyl(Echinocandin C) 2. Deoxypneumocandin A₀ OH H OH OH CH₂—CO—NH₂10,12-Dimethyl- myristoyl 3. Deoxypneumocandin A₁ H H OH OH CH₂—CO—NH₂10,12-Dimethyl- myristoyl 4. Deoxypneumocandin A₂ OH H H H CH₂—CO—NH₂10,12-Dimethyl- myristoyl 5. Deoxypneumocandin B₀ OH H OH OH CH₂—CO—NH₂10,12-Dimethyl- myristoyl 6. Deoxypneumocandin B₂ OH H H H CH₂—CO—NH₂10,12-Dimethyl- myristoyl 7. Deoxypneumocandin C₀ OH H OH OH CH₂—CO—NH₂10,12-Dimethyl- myristoyl 8. Deoxymulundocandin OH H OH OH H 12-Methyltetra- decanoyl

particularly to a Process for the conversion of Mulundocandin (compoundof the formula II

to deoxymulundocandin (compound of the formula III)

which consists of a single step selective reduction of C4-htyr(homotyrosine) hydroxyl group of echinocandins to their monodeoxyanalogues particularly under neutral conditions without priorprotection, deprotection of the equally facile C5-Orn (ornithe) hydroxylgroup and purification of the monodeoxy compound from the crude reactionmixture.

DETAILED DESCRIPTION OF THE INVENTION

The conversion of echinocandins to their monodeoxy analogues byselective reduction at C4-htyr may be effected by hydrogenolysis withRaney nickel in solvents such as methanol, ethanol or dioxane at pH 3-9.Preferably, the selective reduction is carried out by hydrogenolysiswith Raney nickel in ethanol at pH 7 and room temperature in the ratioof 6.8 ml Raney nickel per millimole of mulundocandin.

The monodeoxy compounds of the invention may, for example, be purifiedfrom the crude reaction mixture as follows:

By fractionation using normal phase chromatography (using alumina orsilica gel as stationary phase and eluents such as petroleum ether,ethyl acetate, dichloromethane, chloroform, methanol or combinationthereof), reverse phase chromatography (using reverse silica gel likedimethyloctadecylsilylsilica gel, also called RP-18 ordimethyloctylsilsilica gel also called RP8 as stationary phase andeluents such as water, buffers such as phosphate, acetate, citrate (pH2-8) and organic solvents such as methanol, acetonitrile, acetone,tetrahydrofuran or combination of solvents). gel permeationchromatography—using resins such as SEPHADEX LH-20® (Pharmacia ChemicalIndustries, Sweden), TSKgel Toyopearl HW (TosoHaas, Tosoh Corporation,Japan), in solvents such as methanol, chloroform or ethyl acetate ortheir combination or SEPHADEX G-10® and SEPHADEX G-25® in water; or bycounter-current chromatography using a biphasic eluent system made up oftwo ore more solvents such as water, methanol, ethanol, iso-propanol,n-propanol, tetrahydrofuran, acetone, acetonitrile, methylene chloride,chloroform, ethylacetate, petroleum ether, benzene and toluene. Thesetechniques may be used repeatedly or a combination of the differenttechniques may used. Counter-current chromatography (liquid-liquidchromatography) using a biphasic eluent system ITO coil is preferred forpurification of the compounds of the invention.

The following experimental example is illustrative of the presentinvention but not limitative of the scope thereof.

Example 1

Mulundocandin (220 mg, 2.2 mM) in ethanol (8 ml)) was stirred with 15 mlof W-2 Raney nickel (pH 7) in ethanol (30 ml) for 3 hours at roomtemperature. After standing for 15 minutes the supernatent solution wasdecanted and Raney nickel washed with 3×30 ml. ethanol with stirring andfiltered. Combined ethanolic solutions were concentrated by distillationunder a reduced pressure of 60-70 mm/Hg at 35° C. to obtain 160 mg (75%)of crude deoxymulundocandin as a slightly green solid.

The crude product was purified by liquid-liquid chromatography on ITOcoil using upper layer of CH₂Cl₂:MeOH:n-PrOH:H₂O as the stationary phaseand the lower layer as the mobile phase in an ascending mode. The coils(15+25+215 ml) were connected in series and a flow rate of 0.6 ml/min.at a piston stroke of 60 and pressure 0.5 bars was maintained. Thepurification of deoxymulundocandin was monitored both by bioactivityagainst Candida albicans and Aspergillus niger and by analytical HighPressure Liquid Chromatography (HPLC) [column: (10×0.4 cm+3×0.4 cm)ODS-Hypersil, 10μ; mobile phase: 50:50 CH₃CN:H₂O; flow rate:1 ml/min;Wavelength; 220 nm.) The fractions (4.5 ml each) containingdeoxymulundocandin were combined, concentrated by distillation under areduced presssure of 60-70 mm/Hg at 35° C. and lyophilized to yield puredeoxymulundocandin [65 mg (30% yield)]. Also recovered during the abovepurification of deoxymulundocandin was unreacted mulundocandin in 10%yield.

The semi-synthetic deoxymulundocandin was identical in all respects tothe naturally isolated compound and the physico-chemical data is givenin Table 1.

TABLE 1 Appearance White powder Melting point 170-172° C. [α]_(D) −36.6°(c 0.25, MeOH) HPLC RT 4.42 min FAB-MS (Fast Atom 1014.7 (M + Na)⁺Bombardment mass) ¹H NMR (300 MHz, FIG. 1 of the accompanying drawingsCD₃OD) ¹³C NMR (75 MHz, FIG. 2 of the accompanying drawings CD₃OD)

What is claimed is:
 1. A process for converting mulundocandin of formulaI:

wherein each of W, x, Y and Z is OH, R is H and R′ is12-methyl-tetradecanoyl, to deoxymulundocandin, a compound of formula Iwherein W, Y and Z are each OH, X and R are each H and R′ is12-methyl-tetradecanoyl, this process comprising reducing the C4-htyr(homotyrosine) hydroxyl group of said mulundocandin by mixing saidmulundocandin with Raney Nickel in a solvent selected from the groupconsisting of methanol, ethanol and dioxane at a pH of 3-7 withoutprotecting and then deprotecting the C5-orn (ornithine) hydroxyl group,and then purifying the resulting deoxymulundocandin from the crudereaction mixture.
 2. A process as claimed in claim 1, wherein saidreducing of the C4-htyr (homotyrosine) hydroxyl group is carried out byhydrogenolysis with Raney nickel in ethanol at pH 7 and at roomtemperature.
 3. A process as claimed in claim 2, wherein thehydrogenolysis is carried out in the ratio of 6.8 ml of Raney nickel permillimole of mulundocandin.